title: The Cre/loxP recombination system for production of infectious mouse polyomavirus.
abstract: Murine polyomavirus mutants are frequently produced for experimental as well as therapy purposes. Commonly used methods for preparation of mutant viral genomes from recombinant vectors are laborious and give variable yields and quality. We describe an efficient and reproducible Cre/loxP-mediated recombination system that generates polyomavirus genomes from recombinant plasmid in vivo. We designed and constructed two variants of recombinant vectors containing the wild-type polyomavirus genome flanked by loxP homologous sites. The loxP sites were introduced either into the intronic region of early genes or between the two poly(A) signal sites of convergent transcriptional units. After cotransfection of the recombinant plasmids with the Cre-expressing vector into mouse 3T6 cells, we obtained infectious virus from the genome variant containing loxP site in the intronic region, but we failed to isolate any infectious virus from the viral genome containing loxP site between poly(A) signals. We show that the Cre/loxP-based method of polyomavirus production is simple, expedient, and reproducible and works with satisfactory efficiency.
authors.affiliation: Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague 2, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Hron
authors.forename: Hana
authors.initials: H
authors.lastname: Spanielová
authors.forename: Jiřina
authors.initials: J
authors.lastname: Suchanová
authors.forename: Jitka
authors.initials: J
authors.lastname: Forstová
mesh_terms: D000818:Animals; D002460:Cell Line; D051379:Mice; D008967:Molecular Biology / Q000379:methods; D010957:Plasmids; D011120:Polyomavirus / Q000235:genetics* / Q000254:growth & development*; D011995:Recombination, Genetic; D014773:Virology / Q000379:methods*
keywords: Cre recombination; MPyV; Murine polyomavirus; PAS; Viral genome modification; Virus production; loxP; polyadenylation signal